Manipulating matter at the nanometer scale is important for many electronic, chemical and biological advances (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166-169, 2001). A number of sequencing techniques have been proposed at the micrometer and nanometer scale in response to the human genome project. These techniques have been largely developed to help characterize and understand expression of genes in vivo. A popular technique uses micro arrays and hybridization of cDNA to determine the presence or absence of a particular target gene. A target gene and probe are exposed in solution and bind if relative hybridization sequences match. Hybridization is indicative of the presence of the sequence or target gene. A dye may be employed with the target or probe to then determine existence and efficiency of hybridizations. The technique has been extended for use in determining the presence of single nucleotide polymorphism (SNP'S) in target cDNA. Micro arrays provide the promise of being able to accurately and concurrently screen for a variety of diseases in a particular patient. A few major drawbacks of the micro array technique concerns difficulty in manufacturing as well as the long time for effective hybridizations between probe and target (generally overnight to maintain high specificity). In addition, the large amounts of genomic DNA in a patient would require an inordinate amount of time and work to sequence. Therefore, new techniques are now being explored to more quickly sequence biopolymers. Systems that are on the nanoscale are both effective on resources (limited materials), but also may more closely mimic the high speed processes already present in living cells (i.e. translocation processes). Therefore, nanopore technology has become a fundamental field of interest to molecular biologists and biochemists alike.
It has been demonstrated that a voltage gradient can drive single-stranded biopolymers through a transmembrane channel, or nanopore (See Kasianowicz et al., “Characterization of individual polynucleotide molecules using a membrane channel”, Proc. Natl. Acad. Sci. USA, 93: 13770-13773, 1996). During the translocation process, the extended biopolymer molecule will block a substantial portion of the otherwise open nanopore channel. This blockage leads to a decrease in the ionic current flow of the buffer solution through the nanopore during the biopolymer translocation. The passage of a single biopolymer can be monitored by recording the translocation duration and the blockage current, yielding plots with predictable stochastic sensing patterns. From the uniformly controlled translocation conditions, the lengths of the individual biopolymers can be determined from the translocation time. Furthermore, the differing physical and chemical properties of the individual monomers of the biopolymer strand may generate a measurable and reproducible modulation of the blockage current that allows an identification of the specific monomer sequence of the translocating biopolymer. These initially proposed systems suffer from a number of problems. For instance, some of the proposed systems require the self-assembly of pore forming proteins on membranes (i.e. α-hemolysin). Reproducibility of membranes and systems has been quite problematic. Secondly, commercial products require robustness not present in sensitive systems that require fluctuations of ionic currents for measurements. For these reasons, recent research has focused more on solid-state pore techniques that have an ability for high reproducibility and ease of fabrication. Such techniques as “ion beam sculpting” have shown some promise in fabricating molecular scale holes and nanopores in thin insulating solid-state membranes. These pores have also been effective in localizing molecular-scale electrical junctions and switches (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166-169, 2001).
These techniques have shown similar consistent results and current blockage with double stranded DNA reminiscent of ionic current blockages observed when single stranded DNA are translocated through the channel formed by α-hemolysin in a lipid bilayer. The duration of these blockages have been on the millisecond scale and current reductions have been to 88% of the open-pore value. This is commensurate with translocation of a rod-like molecule whose cross-sectional area is 3-4 nm2 (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166-169, 2001). This methodology, however, suffers from the limitation that only crude measurements of the presence or absence of the translocating polymer can be made. In addition, these systems are incapable of actually determining the primary sequences (order of monomeric units) of the translocating biopolymer.
A second approach has been suggested for detecting a biopolymer translocating a nanopore in a solid-state material such as Si3N4. However, it is well known that the tunneling current has an exponential dependence upon the height and width of the quantum mechanical potential barrier to the tunneling process. This dependence implies an extreme sensitivity to the precise location in the pore of the translocating molecule. Both steric attributes and physical proximity to the tunneling electrode could cause changes in the magnitude of the tunneling current which would be far in excess of the innate differences expected between different base-types under ideal conditions. For this reason, it is difficult to expect the simplest tunneling configurations to have the specificity required to perform sequencing.
Although there are a number of important techniques for nanopore sequencing being developed, there still remains problems regarding getting the actual biopolymer to the nanopore. In addition, there remains the issue of actually threading the biopolymer into the nanopore so that its sequence may be accurately determined. A few techniques have been developed to potentially address this issue. For instance, the application of hydrodynamics and pressure applied to a closed system has been considered for directing biopolymers into nanopore structures. These inventions have a number of limitations not limited to increase in cost and safety. Secondly, some work has focused on use of electric fields to potentially draw the biopolymer through the nanopore structure. A problem with such techniques is that the biopolymer does not easily move through such defined spaces unless it is at first “threaded” into or through the nanopore. Some work has been done on passing biopolymers through fabricated nanopores in membranes of silicon nitride or silicon dioxide. Further, there is the continual need to monitor the position of the biopolymer in the nanopore as well as having the end of the biopolymer enter the nanopore as opposed to the middle of the material. In addition, there is the need to control the motion of the biopolymer in the nanopore as well as to stretch or extend the biopolymer in the nanopore so that it may be correctly and accurately sequenced. These and other problems with the prior art processes and designs are obviated by the present invention. The references cited in this application infra and supra, are hereby incorporated in this application by reference. However, cited references or art are not admitted to be prior art to this application.